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We have investigated the physical, biochemical, and cellular properties of an autologous leukocyte and platelet-rich fibrin patch. This was generated in an automated device from a sample of a patient's blood at the point of care. Using microscopy, cell counting, enzyme-linked immunosorbent assay, antibody arrays, and cell culture assays, we show that the patch is a three-layered membrane comprising a fibrin sheet, a layer of platelets, and a layer of leukocytes. Mean recovery of platelets from the donated blood was 98% (±95%CI 0.8%). Mean levels of platelet-derived growth factor AB, human transforming growth factor beta 1, and vascular endothelial growth factor extracted from the patch were determined as 127 ng (±95% CI 20), 92 ng (±95%CI 17), and 1.35 ng (±95%CI 0.37), respectively. We showed a continued release of PDGF-AB over several days, the rate of which was increased by the addition of chronic wound fluid. By comparison with traditional platelet-rich plasma, differences in immune components were found. The relevance of these findings was assessed by showing a mitogenic and migratory effect on cultured human dermal fibroblasts. Further, we showed that fibrocytes, a cell type important for acute wound healing, could be grown from the patch. The relevance of these findings in relation to the use of the patch for treating recalcitrant wounds is discussed.
Chronic wounds remain a challenge for patients as well as health providers. Treating chronic wounds with autologous growth factors has shown promising but diverse results. The main ways of providing growth factors for the treatment of wounds has been by the use of recombinant PDGF-BB (Regranex gel, Systagenix, Gatwick, United Kingdom) or the generation of platelet-rich preparations, either based on platelet concentrates, platelet-rich plasma (PRP), or recently, platelet-rich fibrin (PRF). PRP-based treatments are performed by activating a PRP suspension with thrombin, typically either bovine (Autologel System, Cytomedix, Gaithersburg, MD) or autologous (Biomet, Warsaw, IN), and/or calcium ions, thereby generating a platelet gel incorporating the activated platelets. Several ways of generating platelet-based treatments for wound treatment have been developed based on either allogenic or autologous material. The latter ranges from simple laboratory instructions using standard equipment to complex systems with dedicated reagents, hardware, and utensils (Vivostat [Allerød, Denmark], GPS [Biomet], and SmartPrep [Harvest Technologies, Plymouth, MA]). Studies comparing blood cell, particularly platelet, recoveries, and growth factor release from the resulting preparations generally show that there is a high diversity in composition between different preparations and within preparations produced by the same device when determined on different donors.4,5
Clinical evidence for the effectiveness of platelet-rich products is limited, mainly due to small uncontrolled studies. However, a recent meta-analysis of published studies found that PRP favors the healing of diabetic foot ulcers. PRF could have several advantages over PRP and other platelet-rich products due to its high fibrin content; these include greater mechanical strength, extended release of growth factors, and the protection of growth factor against proteolysis.7,8 Studies have shown that growth factors derived from platelets interact with the fibrin (vascular endothelial growth factor [VEGF], basic fibroblast growth factor, fibroblast growth factor 2 [FGF-2]). Furthermore, studies show that these interactions could be the basis for protection from proteolytic degradation7,12 and growth factor potentiation. Although existing PRFs are similar to PRP in terms of growth factor contents, only a few clinical studies have been performed in nonhealing wounds.
In order to be clinically relevant, the treatment should have an effect on wound healing and the processes involved, including the formation of granulation tissue. Granulation tissue is mainly formed by the fibroblast and endothelial cells resident in the wound and its surrounding tissue. Additionally, studies have shown that a subpopulation of circulating leukocytes, the fibrocytes, is involved in wound healing. In acute wound healing, these cells migrate to the wound and are involved in several processes important for proper healing. Fibrocytes are described by their hematopoietic origin combined with the synthesis of extracellular matrix proteins (e.g., collagen).18,19
In this study, we characterize a new platelet and leukocyte-rich fibrin patch produced by a two-spin process in a closed sterile system. In contrast to the PRF produced by others (e.g., Dohan et al.), the patch described displays a three-layered structure as well as a high content of platelets and leukocytes. We investigate the structure and ultrastructure of the patch as well as the release of selected growth factors. To relate results to traditional PRP, we compare released growth factors, cytokines, and chemokines by antibody arrays as well as enzyme-linked immunosorbent assay (ELISA) of selected factors. Further, we determine the effect of patch-derived factors on normal human dermal fibroblasts (NHDF) cells and the release of autologous fibrocytes. Finally, we discuss the relevance of these characteristics to its proposed use for the treatment of difficult to heal chronic wounds.
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- Supporting Information
Advances in wound care products in the recent decades have mainly been due to the development of advanced materials able to maintain a wound milieu optimal for the intrinsic wound healing. Despite this effort, there is consensus that nonhealing wounds benefit from treatments actively affecting the wound-healing cascade.
We have characterized a leukocyte and platelet rich patch in regard to its physical as well as biochemical and cellular properties. The three-layered compacted nature of the patch (Figures 1 and 2) differs it from traditional preparations based on autologous blood. This is due to the surprising finding that at high centrifugation speed, no red blood cell coagulum is formed; rather, a coagulum binding only platelets and leukocytes of the buffy coat is generated. By compacting this coagulum by mechanical means as in the Leucopatch Device, a patch with a defined structure is formed.
The donors used in this study represent both males and females (six and four, respectively) as well as both A, B, and O blood types. Despite observations that raise the possibility for a greater tendency for blood clot formation in non-O patients, probably due to differences in Von Willebrand factor plasma half-life, we did not see any difference in coagulation between O and non-O donors nor between male and female.
As it was not feasible to count cells that were incorporated onto the patch, we determined cell recovery by counting cells remaining in the device after removal of the patch. We assessed the remains by microscopy and filtration to assure that it was a homogenous solution that was measurable by an automated (Sysmex) cell counter. As the Sysmex detection system uses an impedance-based detection method that has previously shown to flag platelet clumps with adequate sensitivity, we are confident in the method used. We hypothesize that the high platelet recovery rates as well as the absence of red cells in the patch are due to the complete compaction of red blood cells during the initial hard spin, and this lead to a near complete separation of red blood cells, platelets, and plasma. Upon polymerization, fibrin binds the buffy coat but the smoother compacted red cells are not bound. This theory fits well with the recoveries seen in our study as the cells with the highest specific gravity (SG), neutrophils (SG: approx. 1.08), have the lowest recovery (55%); in contrast, the denser lymphocytes (SG: approx. 1.07) and monocytes (SG: approx. 1.06) show higher recovery rates (77% and 85% respectively). The lower density of the platelets (SG: 1.05) leads to a recovery of 98%. That a near complete equilibrium is reached is confirmed by the layered structure of the produced patch having leukocytes below the platelet layer (Figure 1C). Further, the significant correlation between full blood platelet numbers and the extracted PDGF-AB and VEGF factors supports the high recovery rate seen.
There is a high degree of variation between PRP/PRF systems, and because of variable ways to measure the cell recovery and growth factor release, direct comparisons are difficult. Data reviewed by Mazzucco et al. described nine different systems with platelet recoveries ranging from 17 to 90%, and PDGF-AB release ranging from 33 to 140 ng/mL. By comparison, we found a 98% platelet recovery and an average release of 127 ng PDGF-AB per patch. Calculating the growth factor contents in relation to the number of platelets drawn from the donor has been suggested to enable a better comparison between products. We found 37.1, 27.3, and 0.381 pg per 106 platelets for PDGF-AB, TGF-β, and VEGF, respectively. This corresponds well with growth factors released over 5 hours from Choukrons PRF: PDGF-AB: 27.8, TGF-β: 38.3, and VEGF: 0.55 (recalculated to pg/106).
It is well known that CWF has a strong proteolytic activity and we have previously shown an increased growth factor release from a PRF product under proteolytic conditions. We show that increased PDGF-AB release is seen when the patch is incubated with CWF and that this increase appears to be related with the solubilization of the patch. The continued release of PDGF-AB is in contrast to the instant release seen with thrombin activation of PRP.
To assess the effects of the large numbers of leukocytes included in the patch, we made a direct comparison with simultaneously generated PRP. Comparing PDGF-AB, VEGF, and IL-8 between the two preparations revealed differences as would be expected as platelet and leukocyte concentrations were higher in the patch. The classical PRP method used resulted in an average recovery of 48% platelets into a leukocyte-free PRP. Relating growth factor levels to the platelet numbers in the PRP and patch PDGF-AB release were higher in the PRP. This fits well with the ability to continuously release PDGF-AB from the patch preparation. In contrast, both VEGF (twofold) and IL-8 (70-fold) were increased despite this correction. To relate the levels to clinical application, we calculated the actual levels applied at a certain area. These numbers show approximately threefold, eightfold, and 320-fold increase for PDGF-AB, VEGF, and IL-8, respectively. The highly increased level of IL-8 fits well with the high leukocyte contents. IL-8s are involved in the needed acute response initiating wound healing and have been suggested for the treatment of chronic wounds.
By comparing PRP and patch extracts by antibody arrays, the levels of 10 proteins were found to differ; of these, eight were present at higher levels in the patch extracts. These included growth factors involved in angiogenesis (VEGF and FGF-2) as well as reepithelialization (HGF). Cytokines (IL-8, IL-16, CD40L) and immune modulators (IL-1ra), as well as the soluble pattern recognition molecule, PTX3, which are involved in the body's first line of defense as part of the innate immune response. Surprisingly, the collagenase MMP-8 was present in higher levels in the PRP preparation despite its known production by neutrophils. Finally, leptin was found at higher levels in PRP; leptin has been shown to be involved in keratinocyte proliferation.
In order to assess the biological potential of the growth factors released from the patch, we followed the growth of primary human fibroblasts in response to patch-derived substances. An insert-based assay was set up to prevent the detection of patch-derived cells. The results showed a strong mitogenic effect on the human fibroblasts used, confirming the biological activity of the patch-derived growth factors. We have previously shown a similar effect of both a traditional PRP and the Vivostat PRF. Increased fibroblast migration seen in initial studies confirm the biological effect of both patch and PRP-derived factors, although the effect of PRP seems to be lower than patch at the highest concentrations tested (Supporting Information Figure S2). As leukocyte-derived fibrocytes have been implicated in the healing process, we investigated if these cells could be derived from the patch. We showed that fibrocytes can be grown from the patch. The theoretical benefit of this finding has to be confirmed by further studies.
Acute wounds are known to heal by going through several steps: hemostasis, inflammation, proliferation, and remodeling. Nonhealing wounds are believed to be halted in the inflammatory phase. Studies on acute wounds show that extremely high numbers of neutrophils are present within hours of wounding, but disappear within days, followed by the presence of monocytes and macrophages that transfer the wound to the proliferative phase of wound healing. Although highly complex, recent studies suggest that the apoptosis of neutrophils and its phagocytosis by monocytes/macrophages leads to a decrease in neutrophil migration to the wound and the transfer of the macrophages into a so-called wound-healing phenotype.38,39 Abrogated healing of macrophage-depleted mice confirms the importance of these cells. Other studies have shown weak perivascular infiltrates in diabetic ulcers, despite the up-regulation of attachment molecules known to be involved in the acute inflammation and wound healing. The resulting low-grade chronic inflammation leads to a lack of an acute-focused immune response, essential for the healing cascade to continue. This is in line with a study showing that accelerated healing in mice was associated with an increased infiltration of leukocytes and fibrocytes.
In this study, we show that a leukocytes and platelet rich patch can provide a way of transferring concentrated cells and signals directly to a surface. On that basis, we suggest that the patch described here could be beneficial for the healing of recalcitrant wounds. To investigate this potential, in a relevant setting, clinical testing in humans is needed. A pilot trial on 15 patients, with 16 lower extremity chronic wounds of varying etiologies, has been performed with a positive outcome and a larger multicenter trial on diabetic foot ulcers is currently being performed.